2-hydroxy-9alpha-fluoro pregnenes and pregnadienes



3,063,991 2-HYDROXY-9oc-FLUORO PREGNENES AND PREGNADIENES Louis I.Feldman, Spring Valley, N.Y., Neil E. Rigler,

This invention relates to a microbiological hydroXylation of steroids.More particularly, it relates to the 2 3- hydroxylation of steroids ofthe pregnane series and novel 2 3-hydroxy steroids resulting therefrom.

The compounds prepared by the process of the present invention can beillustrated by the following general formula:

wherein C -C is a divalent radical selected from the group consisting ofCH CH and CH=CH radicals, X is a radical selected from the groupconsisting of (5)0H O and O=O (00H radicals, R is selected from thegroup consisting of hydrogen and lower alkanoyl radicals, Y and Z areselected from the group consisting of hydrogen and halogen atoms and Rand R are the same or different and are hydrogen atoms or lower alkylradicals.

The microbiological process of the present invention is carried outunder aerobic conditions in the presence of a suitable nutrient mediumat a temperature within the range of from about 20 C. to about 40 C. Thesteroid to be Zfi-hydroxylated is added to the nutrient medium andfermentation is carried out with Streptomyces grz'seus. Thetransformation taking place in the reaction medium can be traced bypaper chromatographic assay and is usually complete Within about fourdays. In carrying out the process of the present invention, Streptomycesgriseus (Strain Al) (ATCC No. 13,968) has been found to give goodresults. During the growth of the organism under favorable conditions, ahydroxyl group is introduced into the ZB-position of the steroid ring A.The exact mechanism of this 218-hydroxylation is not known but isbelieved to be an enzymatic reaction.

A suitable nutrient medium for the fermentation of the present inventioncontains a soluble source of carbon, nitrogen and mineral traceelements. Sources of carbon include sugars such as glucose, sucrose,maltose, dextrose, xylose, galactoses and so forth. Also, alcohols suchas glycerol or mannitol, corn starch, etc., organic acids such as citricacid, maleic acid, acetic acid and various natural products containingcarbohydrates such as corn steep liquor, soya bean meal, cotton seedmeal and many available materials which have been used heretofore as asource of carbon in fermentation processes. Usually a variety of theabove carbon sources are used in a medium which gives the best results.Suitable sources of nitrogen in- I anneal: Patented Nov. 13, 1952 eludesome of the above named materials such as corn steep liquor, soya beanmeal, cottonseed meal and the like and various other substances such asbeef extract, casein, yeast, enzymatically digested proteins anddegradation products including peptones, amino acids and many otheravailable proteinaceous materials which have been found to be suitablein supporting the growth of Streptomyces griseus. Inorganic sources ofnitrogen include urea, ammonium salts, nitrates and the like. The lattermay be used in the medium as a source of nitrogen to provide a favorablegrowth medium for the organisms.

The mineral requirements of fermentation are usually supplied in thecrude materials which are often used as sources of carbon and nitrogenor in the Water that is used in the process. However, it is oftenadvisable to supplement the minerals normally present with added amountsto obtain maximum growth. Cations and anions which may be desirable inadded amounts include sodium, potassium, calcium, magnesium, phosphate,sulfate, chloride, cobalt, manganese and various others. The use ofelements such as boron, copper, molybdenum and chromium is oftendesirable.

The growth of the organism takes place under aerobic conditions, andaeration in flasks, for example, can be achieved by agitation on areciprocating or rotary shaker or in bottles or tanks by forcing sterileair through the fermentation mixture. It is desirable that the sterileair be forced through the medium in an amount of from /3 to 2 volumes ofair per volume of medium per minute. Agitation in the bottles orfermenter tanks is provided by a mechanical impeller. While the organismwill grow at temperatures between 5 and 45 C., it is preferable to carryout the process as stated hereinbefore at a temperature of from about 20to about 40 C.

The A steroids of the pregnene series which can be used in the processof the present invention include, for example,

9a-flu0ro-11,B,21-dihydroxy-16a,17a-isopropylidenedioxy- 4-pregnene-3,20-dione; 115,21-dihydroxy-16 17a-isopropylidenedioxy-4- pregn'ene-B,ZO-dione;

9a-fiuoro-l 15.21-dihydroxy-16a, l7a-isopropylidenedioxy-1,4-pregnadiene-3,20-dione and 60:,9oc-difi110IO-l1,6,21-dihydroxy-16u,l7a-isopropylidenedioxy-4-pregnene-3 ,ZO-dione andesters thereof and the like. When using the above steroid substrates inthe fermentation, the products formed are the free alcohols of thesesteroids. It is generally desirable that the steroids be added to thefermentation in solution or in finely divided form. A preferred methodis to dissolve in methanol or other water miscible solvents and add itto the fermentation medium at the desired stage in the process. Althoughthe steroid may precipitate from solution when so added, it is dispensedthroughout the medium as a fine suspension and becomes readily availableto the organism for oxidation. The amount of steroid added to thefermentation may vary considerably,

but it is generally on the order of li to 1 gram per liter of medium. 1

To prepare inocula, 1.0 ml. of washed spore and cell suspension of theStrepotomyccs griseus (Strain A-l) is used toin'oculate 1'00 mlfofsterile medium such as describedinthe'exarnples' hereinafter a 500 ml.flask. The medium is sterilized by auto'claving for 15 minutes at1'5'pounds steam pressure (120 C.). The inoculated flask is incubated at abou t 28 C. on a shaker for about 24. 72 hours. Such inocula may beused to inoculate larger batches of sterile medium in bottles and suchbottle cultures, after fermentation, may be used 'to inoculate largebatches of medium in fermenter tanks.

During'the fermentation process, it may be desirable to add;anti-foaming agents such as silicones, glyceride oils and the like.These compounds are added from time to time in the amounts needed. Inthe process of the present invention, the 100] ml. batches of inoculatedmedium in 500 ml. flasks are usually incubated for a period of 1,6 to 40hours at a temperature of about 28 C. this point, mg. of substratesteroid dissolved in 1 ml. of methanol is. added to each flask and thefermentation continued, at about 28 C. The fermentation is allowed toproceed for a period of time long enough to achieve maximum conversionof the'steriod. substrate to the corresponding 2B-hydroxysteroid This.period may vary fromfseveral hours to 144 hours or longer.

At the conclusion'o-f. the fermentation process the 2,8- hydroxylate'dsteroid is. recovered from the fermentation medium by the followingprocedure. The contents of the fermentation tube are extracted withthree volumes of ethyl acetate. The ethyl. acetate phase is evaporatedto dryness and the residue dissolved in'an' appropriate volume of amixture consisting of a 1:1 ratio of watersaturated ethyl acetate andmethanol. This solution is used, for characterization of steroid contentas described hereinafter. i

In large scale fermentations, the crude product or products may berecovered from the fermentation beer by simple solvent extraction usinga water immiscible solvent such as, for example, chlorinatedhydrocarbons, alcohols, esters, ketones and so forth. Furtherpurifications and separations of the steroid products from extractionsmay be accomplished by 'methods well known to those skilled in the art'.Separation and purification of steroid mixtures often'require the use ofchromatography.

The 2fi-hydro'xylating process of the present invention is useful forpreparing products that are active glucocorticoids and can be employedas chemotherapeutic agents useful in the same manner as cortisone orliydrocortisone for the treatment of arthritis, bursitis, burns and thelike. The compounds prepared by the process of the present inventioncontain a .2-hydroxly group which enhanceswater solubility making thecompounding of pharmaceutical preparations less diffi'cult. Also, theenhanced Water solubility can favorably affect the in vivo activities of1601,176Fisopropylidenedioxy steroids in humans.

The, following examples illustrate in detail the preparation of2-hydroxy steroidsj from the corresponding steriods of the pregnaneseries; 1

EXAMPLE I Que hundred. ml. of sterile medium. (No. 60) consisting of,soybean'meal, 0.22%, corn steep liquor, 0.3%, glucose, 1.0%, yeastextract, 0.25%, ammonium biphosphate,0.3%, and calcium carbonate, 0.25%,adjusted'to pH 7.0.with sodium hydroxide in a 500 ml. Erlenmeyer flaskis inoculated with 1 ml. of a 72 hour mycelial growth of Streptomycesgriseus strain A-l (ATCC No. 13968). The flask is placed on areciprocating shaker at 28 C. for 16 hours. At this time, 10 mg. of9a-fluoro- 11,8,21 dihydroxy 16a,17a-isopropylidenedioxy-4-preg- 4nene-3,20-dione dissolved in 1 ml. of methanol is added. Shaking iscontinued for 72 hours at which time paper chromatographic assays showa25% yield of 9a-fiuoro- 2 3,115,2l-trihydroxy-16a,17 tisopropylidenedioxy-A-pregnene-3,20-dione.

EXAMPLE 11 Preparation of 9a-Flu0r0-2,8,11,B-21-Trihydr0xy-16a,17a:-Isopropylidenedi0xy4-Pregnene-3,ZO-Dione The pH of the medium consistingof soybean meal, 0.22%, dextrose, 1.0%, corn steep liquor, 0.3%, yeastextract, 0.25% and ammonium biphosphate, 0.3 is adjusted to about 7.0before adding the calcuim carbonate 0.25%. The inoculum is grown for 72hours on a reciprocating shaker. At the end of this period of time, theinoculurn is transferred to a five gallon bottle containing 12' litersofthe above medium. After a further 16 hours, 2.5. g, of 9-fiuo-ro-11.5,21-dihydro5ry-1604,171 isopr'opylidenedioxy 4pregnene-3,20-dione dissolved in ml. of methanol is added to the bottle.Conversion is allowed to continue for 81 hours, at which time the bottleis harvested.

Two such bottles are fermented each with 2.5 g. of steroid. The combinedbottles are extracted with an equal volume (22 liters) of ethyl acetateand then filtered. The ethyl acetate phase is separated and the freshcake mixed with 10 liters of ethyl acetate. The. filtrate is extractedtwice more with ethyl acetate, 22 liters each time. The combined ethylacetate extracts are concentrated to a residue. 7

The residue is treated with 1.5 liters of a solution of 80 parts ofmethanol and 20 parts water by volume. The soluble portion is extractedtwice with 500 ml portions of carbon tetrachloride to remove oilysubstances The carbon tetrachloride extract is discarded. The methanolicsolution is concentrated to a residue under reduced pressure andchromatographed in a column of 600 g. of diatomaceous earth (Celite545), moistened with the lower phase of a mixture of 1 part water, 5parts dioxane and 6 parts cyclohexane. The column is developed withupper phase of the same system and, the peak at 3.8 column retentionvolumesiis collected and concentrated to dryness under reduced pressure.The residue is dissolved in acetoneand' the solution allowed to'standuntil crystals formed. The crystals thus obtained. are recrystallizedfrom acetone and have a melting poiht of 260-261 [0:] +129 in methanol,V

max.

9. 15 (polyhydroxy A -3,2Q-dione). On the basis of the large change inmolecular rotation compared with thcstarting.

(Strain A- 1) for hours, Following completion ofthe fermentation, thedesired product is, recovered as describedin EXampleII.

EXAMPLE 1v Preparation of 9ot-FlZl0lO-ZJ1,3,2]-TrihydVOJty-16a,17a-Isopropylidenedioxy-1,4-Preg11adieIze-3,20-Di0ne To sterile fermentationmedium No. 60 is added 9afluoro 11,8,21dihydroxy-l6a,l7aisopropylidenedioxy- -p g dienc-3,20-dione and thefermentation carried EXAMPLE V Process of Preparing 604,90: Difluoro25,113,21-Trihydr xy 1605,170: Isopropylidenedioxy 4 Pregnene-3,20-D1'0ne This product is obtained by the fermentation of6a,9adifiuoro-11,8,21 dihydroxy 16a,17ocisopropylidenedioxy-4-pregnene-3,ZO-dione in sterile medium No. 60 withStreptomyces griseus (Strain A1) for 144 'hours. The product is isolatedfrom the fermentation medium as described in Example II.

We claim:

1. The compound 9u-fluoro-2,11,3,21-trihydroXy-16u,17a-isopropylidenedioxy-1,4-pregnadiene-3,20-di0ne.

2. The compound 6a,9ot-difluoro-2,8,1 fl,21-trihydroxy-16a,17u-isopropylidenedioxy-4-pregnene-3,20-dione.

References Cited in the file of this patent UNITED STATES PATENTSInhoffen Apr. 28, 1942 Kaufmann et a1. Sept. 19, 1949 Fried July 3, 1956Wettstein Jan. 22, 1957 Miramontes Mar. 17, 1959 Julian May 19, 1959Greenspan et al. Jan. 17, 1961 OTHER REFERENCES

1. THE COMPOUND 9A-FLUORO-2,116,21-TRIHYDROXY-16A,17A-ISOPROPYLIDENEDIOXY-1,4-PREGNADIENE-3,20-DIONE.